PTI Research develops novel fluorescent probes for biomedical applications. Preliminary data has shown that PTIR271, a fluorescent dye well matched to the 647nm line of the Kr-Ar laser line of the confocal microscope has superior diffusion and imaging properties for neuronal profiling in fixed tissue compared to other commercially available dyes. In combination with two other commercially available dyes, PKH2 and PKH26, which are suboptimal for neuronal profiling due to slow diffusion rate and suboptimal excitation respectively, the potential for triple labeling of neuronal profiling has been demonstrated but improvements are needed. In SBIR Phase I, we will standardize test systems in fixed tissue to be used to evaluate new dyes, synthesize a series of PTIR271 analogs to obtain an understanding of structural features of the molecule required for useful diffusion rates of > 2mm/hr in fixed tissue and extend the number of dyes useful for neuronal imaging studies by developing compounds with absorbance's matched to all the major excitation lines of the Kr-Ar laser in confocal microscopes. SBIR Phase I specific aims are to: 1) Standardize test systems for evaluation of new dyes and complete evaluation of PTIR271 (Creighton University); 2) Synthesize PTIR271 analogs to develop SAR information (PTIR); 3) to 6) Create dyes which absorb at 568nm, 488nm, 752nm and 514nm respectively, and are suitable for multicolor neuronal imaging studies (PTIR); 7) Create a dye with a Stokes shift of >50nm which is suitable for multicolor neuronal imaging studies (PTIR); and 8) Create test kits to market each dye to the research community (PTIR). Feasibility will be demonstrated if a set of at least 3 fluorescent lipophilic dyes can be identified for which: . All members of the set diffuse at rates of no less than 2 mm/24 hour with CVs no greater than 10 percent; . Largest and smallest relative dye diffusibility (RDD) values within the set differ by less than or equal to 25 percent; . No member gives S/N >2X background in the filter windows used for detection of other set members; . Relative transcellular labeling (RTL) values for all set members are no greater than the commercial reference dye, Dil.